Autophagy is a tightly regulated intracellular degradation and recycling process that is crucial for cellular homeostasis and adaptation to diverse cancer-relevant stresses. In cancer, current research suggests that autophagy promotes the survival and metabolic fitness of tumors during stress and serves as a resistance pathway during chemotherapy. This has generated significant interest in autophagy inhibition as a therapeutic strategy in cancer; indeed, anti-malarials such as hydroxychloroquine (HCQ) are currently being repurposed as autophagy inhibitors in numerous clinical oncology trials, largely due to their long history of use in humans and well-established toxicity profiles. Despite this enthusiasm, we have much to learn about the long-term consequences of autophagy inhibition in cancer therapy. An important unanswered question is how autophagy impacts metastasis, a principal cause of cancer mortality. The functions of autophagy during metastasis have not been rigorously tested in vivo and our preliminary studies in mouse mammary cancer models indicate that autophagy inhibition in tumor cells results in increased, not decreased, metastasis. These unexpected findings raise the possibility that pharmacological autophagy inhibitors like HCQ may harbor long-term risks by enhancing metastasis in certain cancer patients. In addition, we have obtained preliminary evidence that two mediators of selective autophagy, p62/SQSTM1 and NBR1, direct how the autophagy pathway influences metastasis and tumor cell proliferation during mammary cancer progression and response to therapy. Based on this preliminary data, this proposal seeks to precisely define the stage at which autophagy suppresses mammary cancer metastasis in vivo and to dissect the mechanisms by which autophagy deficiency promotes the metastatic phenotype. We will employ established immmunocompetent mammary cancer models to scrutinize the functions of the autophagy pathway during metastatic progression in vivo. In Aim 1, we will determine the stage-specific effects of autophagy in suppressing metastatic seeding and colonization. In Aim 2, we will determine how p62/SQSTM1 accumulation in autophagy deficient cells impacts metastatic outgrowth. In Aim 3, we will determine the role of NBR1 in adhesion-mediated signaling and metastasis. Overall, these studies provide unique conceptual insight into the role of autophagy during metastasis.